PROJECT SUMMARY EZH2 (Enhancer of Zeste Homolog 2) has a unique, complex and multifaceted link to cancer, mainly because of the fundamental role that the epigenome plays in all major cellular functions. EZH2 is the catalytic component of the Polycomb Repressive Complex group 2 (PRC2), which establishes the repressive epigenetic mark histone 3 lysine 27 trimethylation (H3K27me3), resulting in silencing of gene expression. EZH2 was originally thought to be a tumor-suppressor since loss-of-function events were observed in Myelodysplastic syndrome (MDS), Acute Myeloid Leukemia (AML) and T-ALL. However, recent next-generation sequencing studies have also revealed activating point mutations and amplifications of EZH2 in other cancers, suggesting that it also functions as an oncogene. Specifically, recurrent hyperactive, gain-of-function mutations in EZH2 (e.g. tyrosine residue 641) have been identified in 25% of germinal center diffuse large B cell lymphoma (GC- DLBCL), 15% of follicular lymphomas and 5% of melanoma and sarcoma. Somatic EZH2 amplifications or overexpression also occur in melanoma, breast, non-small cell lung, prostate, uterine and bladder cancer. The role of EZH2 in cancer therefore appears to be cell type-dependent. Using a faithful genetically engineered mouse model of the Y641 mutations, we recently demonstrated that these mutations were capable of driving formation of both lymphoma and melanoma, as in humans. However, several striking observations were inconsistent with the current understanding of the biology of EZH2. Although expression of Ezh2Y641F increased abundance of global H3K27me3, it does not monotonically increase H3K27me3, but rather redistributes the H3K27me3 mark across the genome with complex effects on transcription. These data suggest that the Y641F mutations are not merely hypermorphic, as previously thought, but represent a new class of EZH2 perturbations, exhibiting neomorphic behaviors, with unknown properties and downstream effects. The role of EZH2 during cancer progression is therefore dependent and complicated by cancer cell of origin and it not fully understood. Several paradigm shifts have thus occurred with regards to the biology of EZH2 and gaps in knowledge remain concerning the oncogenic activity of EZH2 and other chromatin modifying proteins. In this proposal, using molecular, genetic and pharmacological methods, I will investigate (1) How Ezh2Y641F results in redistribution of H3K27me3 and the effects of such chromatin re-organization on the rest of the epigenome, (2) How EZH2Y641F-targets, such as Interferon (IFN) signaling, contribute towards its oncogenic activity, and (3) The mechanistic relationship between EZH2Y641F and MYC in B cell lymphoma and the therapeutic consequences of this interaction. Completion of these aims will enhance our understanding of the role of EZH2 during cancer progression, and how regulation of the global chromatin structure contributes towards malignant transformation in lymphocytes. It will also identify other genetic and molecular pathways that could be targeted or utilized for future and more precise therapeutic strategies that would benefit not only patients with EZH2 mutations, but also patients with genetic aberrations in other chromatin modifiers.